3-Hydroxybutyrate Promotes Myoblast Proliferation and Differentiation through Energy Metabolism and GPR109a-Mediated Ca²⁺-NFAT Signaling Pathways

Skeletal muscle wasting is a critical clinical problem associated with several diseases that significantly impair patient outcomes due to the progressive loss of muscle mass and function. This study explores the potential of 3-hydroxybutyrate (3-HB) as a therapeutic agent to counteract muscle atrophy by promoting the proliferation and differentiation of C2C12 myoblasts. Using nuclear magnetic resonance (NMR)-based metabolomics analysis, we uncover the underlying mechanisms by which 3-HB exerts its effects. Our findings demonstrate that 3-HB exerts its effects through two distinct mechanisms: as a metabolic substrate and as a signaling molecule. As a metabolic substrate, 3-HB enhances myoblast energy efficiency by stimulating the expression of G protein-coupled receptor 109a (GPR109a), which subsequently upregulates the 3-HB transporters MCT1 and CD147, the utilization enzyme OXCT1, and phosphorylated AMPK, thereby increasing ATP production. As a signaling molecule, 3-HB activates GPR109a, promoting calcium influx, improving calcium homeostasis, and increasing the expression of Ca²⁺-related proteins such as CAMKK2. This signaling cascade activates calcineurin (CaN), facilitating NFAT translocation to the nucleus and gene expression that drives myoblast proliferation and differentiation. By elucidating the dual regulatory roles of 3-HB in energy metabolism and cellular signaling, this study not only advances our understanding of muscle physiology but also highlights the potential of 3-HB as a novel therapeutic approach for the prevention or treatment of skeletal muscle atrophy.